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PENGENALAN
Protein mkn adalah kompleks Berat molekular yg. berbeza : 5,000 hingga > 1 juta Daltons H,C,N,O,S 20 -asid amino bentuk blok protein Kandungan N dlm mkn : 13.4 - 19.1%
INTRODUCTION
Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. Thus, protein have different molecular structures, nutritional attributes and physiochemical properties.
INTRODUCTION
Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize.
Ikatan peptida
INTRODUCTION
Classification of protein based on composition, structure, biologikal function, solubility. Eg : Simple protein amino acid. Conjugate protein non amino acid component. Denaturation of protein - heat, acid , alkali, organic solvent and detergent. Function and solubility of protein change as well.
INTRODUCTION
Protein : komponen terbanyak dalam sel jalankan fungsi biologikal. Fungsi utama protein:
1. Sumbang tenaga dan nutrien penting 2. Beri ciri fizikokimia kualiti dan atribut sensori 3. Fungsi biologikal in vivo
Enzim Hormon antibodi
INTRODUCTION
Analysis protein is complex food component that shows same physicochemicals as protein.
Eg : Nitrogen non protein free amino acid, small peptide, nucleic acid, fosfolipid, uric acid, urea
INTRODUCTION
Total nitrogen in food consisted of mainly protein & non protein containing nitrogen.
3.
Determination of nitrogen total Determination of colorimetric and determination of protein total quantitative. Determination protein concentration using spectrophotometry
Kjeldahl method
His laboratory technique for nitrogen and protein analysis is still the universally accepted method for this analysis. Other methods claim to be faster and more efficient, but cannot cope with the variety of sizes or conditions of samples than Johan Kjeldahl's original method. Kjeldahl equipment is used extensively all over the world.
Kjeldahl
Kjehdahl method
A food is digested with a strong - releases nitrogen which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food. The same basic approach is still used today, although a number of improvements have been made to speed up the process and to obtain more accurate measurements It is usually considered to be the standard method of determining protein concentration.
Kjehdahl method
Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications. The Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and titration.
1. Digestion
Sample + sulfuric acid + catalyst (anhydrous sodium sulfate ) + copper Heat
1. Digestion
Digestion converts any nitrogen (N) in the food and others in the form of nitrates or nitrites) into ammonia. Ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42). N(food) (NH4)2SO4
2. Neutralization
Product of digestion + H20 + addition of NaOH) NH4 to NH3
(NH4)2SO4 + 2 NaOH 2NH3 + 2H2O + Na2SO4
3. Distillation
The ammonia gas goes into the receiving flask - which contains an excess of boric acid. The low pH of the solution converts ammonia gas into the ammonium ion and converts the boric acid to the borate ion.
4. Titration
The nitrogen content is then estimated by titration of the ammonium borate by H2SO4 or HCl
H2BO3- + H+
H3BO3
5. Calculation
Protein kasar dikira berdasarkan kandungan nitrogen (dari amaun ion ammonia) X faktor protein
Calculation
The following equation can be used to determine the nitrogen concentration of a sample that weighs m grams using a xM HCl acid solution for the titration:
Where vs and vb - titration volumes of the sample and blank 14g - molecular weight of nitrogen N.
Calculation
Once the nitrogen content has been determined it is converted to a protein content using the appropriate conversion factor:
%Protein = %N
X Protein factor
Faktor protein
Campuran protein secara am mengandungi 16% Penentuan dari kandungan nitrogen perlu X faktor protein iaitu 6.25(100/16).
Faktor Protein
Sumber protein Sumber haiwan Susu Nasi Gandum Kacang soya Kacang Tepung Bijirin lain Faktor protein 6.25 6.38 5.95 5.83 5.70 4.6 5.70 6.25
Kaedah Kjehdahl
Prosedur Kjehdahl yg asal AOAC 955.04. Jenis automasi, semiautomasi telah dikembangkan dlm kaedah AOAC 976.06, 976.05 dan 960.52
Penyediaan sampel Sampel dihomogen. Penghadaman Sampel + H2SO4 + CuSO4 + K2SO4 dipanaskan Hasil penghadaman menjadi clear Ammonium sulfat (NH4)2SO4 terbentuk
Penghadam kjeltec
Prosedur Kjehdahl guna semi auto Kjeltec Peneutralan dan penyulingan Hasil penghadaman dicairkan dengan air suling. Sodium thiosulfat ditambah untuk neutralkan asid sulfurik (NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O
H3BO3
Pengiraan
%N=
Isipadu HCl (sampel-blank) N HCl 14 100 Berat sampel 1000
Kelemahan
Kos tinggi Bukan pengukuran nilai protein sebenar Faktor penukaran berbeza diperlukan utk protein yang berbeza
Kaedah Spektrofotometri
Penentuan kepekatan protein
INTRODUCTION
A number of methods have been devised to measure protein concentration, which are based on UV-visible spectroscopy. Kaedah paling sesuai untuk protein tulen. The basic principle behind each of these tests is similar.
A calibration curve of absorbance (or turbidity) versus protein concentration is prepared using a series of protein solutions of known concentration. The absorbance (or turbidity) of the solution being analyzed is then measured at the same wavelength, and its protein concentration determined from the calibration curve
Suitable for
Natural protein system (milk and meat products) Protein is extract in alkali.
Kaedah Biuret
(540-560 nm)
Principal : A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions. The biuret reagent, which contains all the chemicals required to carry out the analysis, can be purchased commercially Left for15 min before reading at 540 nm Kalibrasi: Lengkuk piawai berdasarkan penentuan protein tulen (BSA)
Kaedah Biuret
Digunakan untuk menganalisa protein dalam bijirin, daging, protein kacang soya, makanan ternakan
Kaedah Biuret
Advantages
no interference from materials that adsorb at lower wavelengths less sensitive to protein type because it utilizes absorption involving peptide bonds that are common to all proteins, rather than specific side groups. Not sensitive to component non protein cheap
Kaedah Biuret
Disadvantages Tidak berapa sensitif berbanding kaedah Lowry Keamatan warna berbeza Pelbagai warna muncul jika kandungan lemak dan karbohidrat tinggi
1. 2. 3.
Kaedah Lowry
Kaedah Lowry
The Lowry method combines the biuret reagent with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. . This gives a bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity required. There is a small peak around 500 nm that can be used to determine high protein concentrations and a large peak around 750 nm that can be used to determine low protein concentrations.
Kaedah Lowry
This method is more sensitive to low concentrations of proteins than the biuret method. Less affected by the turbidity of the samples. More specific than most other method
Kaedah Bradford
Pewarna *Coomassie Brilliant Blue G250 (reagen bradford) + PROTEIN
Kemerahan kepada Kebiruan 465 nm kepada 595 nm
Kaedah Bradford
Prosedur
Pewarna* dilarutkan dlm ethanol dan asid fosforik Pewarna* ditambah kepada piawai BSA dan sampel Dibaca pd 595 nm berbanding blank Kepekatan protein dlm sampel ditentukan dari lengkuk piawai BSA
LENGKUK PIAWAI
Kaedah Bradford
Kelebihan
Pantas (2 min) Hasil yang sama berulangkali 7 X > sensitif dari kaedah Lowry Tiada gangguan dari kation spt K+ Na+ Mg2+ X gangguan dari polifenol dan karbohidrat
Kaedah Bradford
Kekurangan
Kompleks pewarna-protein terikat kpd kuvet kuarza. Guna gelas atau plastik Warna berubah ikut jenis protein
PERBANDINGAN KAEDAH